The field of this invention is arrays having associated modified oligonucleotides and/or polynucleotides, methods of producing such arrays, and uses thereof.
Arrays of binding agents, such as oligonucleotides and polynucleotides, have become an increasingly important tool in the biotechnology industry and related fields. These arrays, in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like. One important use of arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
In methods of differential gene expression, arrays find use by serving as a substrate with associated binding fragments such as oligonucleotides. Nucleic acid sequences are obtained from analogous cells, tissues or organs of a healthy and diseased organism, and hybridized to the immobilized set of binding fragments associated with the array. Differences between the resultant hybridization patterns are then detected-and related to differences in gene expression in the two sources.
A variety of different array technologies have been developed in order to meet the growing need of the biotechnology industry. Despite the wide variety of array technologies currently in preparation or available on the market, there is currently no technique for distinguishing the type of binding partner that recognizes a probe sequence on an array (i.e. the ability to distinguish the difference between a target RNA and target DNA sequence). Such an ability would, for example, allow the detection of RNA transcription directly from a cell or tissue without separating the RNA from the DNA. In addition, it would allow the detection of an amplification of specific DNA sequences, e.g., the amplification of an oncogene in a cancer.
There is thus a need in the art for an array that can distinguish between the presence of an RNA molecule and a DNA molecule having identical sequences.
The present invention provides arrays having associated oligonucleotide and/or polynucleotides with modified structures that confer preferential binding to either RNA or DNA relative to the other methods of making such arrays, and assays for using such arrays. The arrays of the present invention have compositions composed of individual polymers that bind selectively to DNA, compositions of individual polymers that bind selectively to RNA, or preferably compositions of each within a single array. The invention comprises an array device comprised of a support surface and polymer molecules bound to the support surface. The polymer molecules are not naturally occurring oligonucleotides or polynucleotides, but rather have modified internucleoside and/or ribose structures with bases attached in the desired sequential positioning and the desired spacing between the bases. The specific modifications allow not just the identification of a particular sequence, but also the identification of the particular type of nucleic acid (i.e. DNA or RNA) having the complementary sequence. Although an array of the invention will have modified oligonucleotides therein arrays of the invention do not exclude the presence of natural oligonucleotides which might also be present to provide comparative information.
In one embodiment, the modified, stably associated polymers of the arrays of the invention preferably recognize RNA. For example, compositions of the arrays have multiple polymers having at least one 2xe2x80x2-5xe2x80x2 internucleoside linkage. In a preferred embodiment, the modified polymers of the compositions of the array are comprised of monomer units with 2xe2x80x2-5xe2x80x2 internucleoside linkages have a plurality of monomer units in the polymer with a 3xe2x80x2-substitution (e.g, a 3xe2x80x2-O-methyl substitution).
In another embodiment, the modified, stably associated polymers of the invention selectively recognize DNA. For example, polymers having at least one p-ethoxy backbone linkage internucleoside linkage bind preferentially to DNA relative to a corresponding RNA sequence.
In a particularly preferred embodiment, the array of the invention is composed of both modified polymer compositions that selectively bind to RNA relative to DNA and modified polymers that selectively bind to DNA relative to RNA. In a particularly preferred embodiment, a single array can have a composition that selectively binds to RNA relative to a corresponding DNA sequence and a composition that selectively binds to DNA relative to a corresponding RNA sequence where the polymers of both of these compositions have the same (meaning corresponding) base sequence for hybridization. This allows a direct comparison of the binding of the two compositions, and will allow a direct determination of the nature of the nucleic acids present in a sample. The array may further comprise corresponding DNA and/or RNA sequences which are not modified.
The compositions of the arrays of the invention are also preferably end-blocked to provide nuclease resistance to the compositions of the array. This allows the arrays to be directly exposed to biological samples which contain nucleases without disrupting the integrity of the array compositions. In addition, nucleases can be used to digest the test substrate binding agent, freeing the associated binding agents for further use. The chemical modification may be on the 5xe2x80x2 end for modified oligonucleotides and/or polynucleotides attached to a substrate at the 3xe2x80x2 end, or alternatively the chemical modification may be on the 3xe2x80x2 end for modified oligonucleotides and/or polynucleotides attached to a substrate at the 5xe2x80x2 end. The associated modified oligonucleotides and/or polynucleotides remain unaffected as to the binding capacity of the associated oligonucleotides. The end block can be placed on modified and/or unmodified sequences on the array.
These arrays also offer the significant advantage that the individual chip can be tested for efficacy and/or quality prior to use with a test sample, which is particularly helpful if the amount of test sample is limited or if the array is being used as a medical device and must comply with FDA quality control requirements.
The invention provides an array that is composed of multiple beads each having associated modified oligonucleotides and/or polynucleotides. Preferably, these beads have a density greater than water and can be centrifuged out of solution or are magnetic to allow the identification and isolation of particular nucleic acid species.
The present invention further provides an assay using the arrays of the invention to determine physiological responses such as gene expression, where the response is determined by the hybridization pattern of the array after exposure to test samples. The test samples may be mRNA, cDNA, whole cell extracts, and the like.
It is an advantage of the associated modified oligonucleotides and/or polynucleotides of the arrays of the invention that the chemical modifications enhance specific chemical binding interactions, e.g., display a differential affinity for RNA or DNA. This allows an array not only to identify the presence of a nucleic acid having a specific sequence in a sample, but also to recognize whether the complementary nucleic acid is RNA or DNA.
It is an advantage of the associated modified oligonucleotides and/or polynucleotides of the invention that the acid stable modifications confer an improved stability on the modified oligonucleotides and/or polynucleotides in an acidic environment (e.g., as low as pH of 1 to 2).
It is another advantage of the associated modified oligonucleotides and/or polynucleotides of the invention that they bind with specificity to specific test nucleic acids.
It is an object of the invention that the modified oligonucleotides and/or polynucleotides can be used in a variety of array applications, such as identification of new genes, determination of expression levels, diagnosis of disease, and the like.
These and other objects, advantages, and features of the invention will become apparent to those skilled in the art upon reading the details of the modified oligonucleotides and/or polynucleotides and uses thereof as more fully described below.